Knife-Edge Scanning Microscopy
Typical microvascular studies examine the number of vessels in a 2D image that corresponds to the viewable area of the slice obtained through traditional histological techniques. Direct comparison to vascular metrics in other studies is challenging because of the lack of standardization of these metrics in the fi eld, both for 2D and volumetric analysis [ 12 , 13 ]. However, a few measurements are commonly used across studies of microvasculature. Here we examine vessel density per unit volume, the ratio between vessel surface area and volume, and vessel segment tortuosity. T ese metrics provide a comparison to prior vascular studies in both 2D and 3D. Because of the non-Gaussian distributions of both volumetric and vectorized measure- ments, a non-parametric Wilcoxon rank-sum test was used to compare signifi cant diff erences between distri- butions in the two regions.
Figure 7 : Image processing steps for grayscale KESM data. (A) First state of acquired image data saved as slices with metadata containing position coordinates as well as camera capture data. (B) An ROI is selected within a bounding cuboid. (C) Known mechanical artifacts in KESM are removed, such as uneven illumination or mechanical limitations in cutting physics (“chatter”). (D) Object classifi cation is performed via 3D object segmentation. Here, blood vessels are identifi ed based on intensity, distance, and morphological criteria. (E) Raster images are transformed into a graph-based representation of the vascular network. (F) The 2D raster images are stacked to create a 3D rendering of the tissue, with typical ROIs in the hundreds of gigabytes range.
compared for each cubic volume ( Figure 8 C, 8 D). Volumes from a mouse brain perfused with India ink were sliced sagittally with a 5 µm thickness in the Z direction, giving a voxel size of 0.7 × 0.7 × 5 µm/voxel. Slices were taken sagittally from the medial to lateral aspect. Each cubic region (512 × 512 × 72 voxels) represents 0.046 mm 3 of brain tissue. T e fractional volume of observed microvasculature (vascular voxel count/ total voxel count) in the forebrain was 1.07%, compared with 3.18% in the cerebellum.
18
We compare vascular density (number of vessels/mm 2 ) between 2D faces of the forebrain and cerebellum volumes, aligned across the Z axis of the volume ( Figure 9A ). Each comparison along the Z axis is equivalent to the vessel density examined in single images in other studies [ 13 ]. Note how the density changes depending on the depth of the 2D face images, indicating the potential bias in the reduced sampling of 2D faces typically performed with other microscopy methods. T e mean vascular density per face was found to be signifi cantly higher in the cerebellum than the forebrain (Wilcoxon rank-sum, Z = −9.40, p < 1e-21) ( Figure 9C ). A high degree of variability in vascular density between 2D faces for both regions was discovered, with a range
spanning from 150 to 200 vessels per mm 2 over a Z depth of 360 µm (72 slices), indicating the importance of identifi cation and verifi cation of structures across all three dimensions of tissue analysis (compare to Figure 9A ).
Another important metric in microvasculature is the surface area to volume ratio, which directly aff ects the diff usion of oxygen and other molecules into the surrounding tissue. T e forebrain region shows a signifi cantly higher surface area to volume ratio (Wilcoxon rank-sum, Z = 9.81, p < 1e-21)
www.microscopy-today.com • 2017 July
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